Stabilization of enzymes is necessary for the long term storage and utilization in many biochemical and biotechnological processes. Enzymes have been isolated from thermophilic organisms which are stable to denaturation by heat. However, even these highly thermostable enzymes may be inactivated by chemical agents, proteases, or environmental modifications. The utilization of thermostable and other enzymes often requires the concomitant use of denaturing conditions including highly elevated temperatures, aqueous environments with sub-optimal concentrations of cofactors and substrates, and a pH that is suboptimal for maximum enzyme stability.
Many stabilization techniques are known. These techniques include immobilization of the enzyme on solid substrates, chemical modification of the enzyme, genetic engineering of the enzyme and the addition of stabilizing additives. Surfactants are one group of additives that have been shown to stabilize enzymes. Surfactants are surface active compounds that stabilize the interface between the active form of the enzyme, and the liquid environment in which they are contained.
For example, non-ionic detergents have been variously shown to increase the solution stability of various proteins with enzymatic activity (e.g., cAMP-dependent protein kinase, tyrosine hydroxylase, nitric oxide synthase, tryptophan hydroxylase and a sweet potato beta-amylase). Additionally, non-ionic detergents such as TRITON X-100 and Tween 20 have been shown to stabilize the activity of DNA polymerases (See, e.g., Biochem., 14: 789-95 [1975]). European Patent Application 776,970 A1, incorporated herein by reference, discloses the use of non-ionic detergents including polyoxyethylated sorbitan monolaurate (Tween 20) and ethoxylated alky phenol (NP-40) to stabilize the activity of Taq thermostable DNA polymerase.
Low concentrations of the anionic detergent sodium dodecyl sulfate (SDS) have been shown to stabilize enzyme activity. However, due to the possibility of cooperative binding if the optimal concentration of SDS is exceeded in solution, the use of SDS in protein stabilization is limited. It is known, however, that many cationic detergents bind less strongly to proteins than strong anionic detergents such as SDS (See e.g., Nozaki et.al., J. Biol. Chem., 249:4452-59 [1974]). Furthermore, most proteins have fewer cationic binding sites than anionic binding sites.
The utility of enzymes such as DNA polymerases often is limited by the stability of the polymerase in solution. Thus, there is need for additives which improve the stability of enzymes in solution, particularly those additives which improve stability as well as avoid the drawbacks of currently used surfactants.